EP1866413A2 - Micrornas and related nucleic acids - Google Patents
Micrornas and related nucleic acidsInfo
- Publication number
- EP1866413A2 EP1866413A2 EP06744785A EP06744785A EP1866413A2 EP 1866413 A2 EP1866413 A2 EP 1866413A2 EP 06744785 A EP06744785 A EP 06744785A EP 06744785 A EP06744785 A EP 06744785A EP 1866413 A2 EP1866413 A2 EP 1866413A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- nucleic acid
- mirna
- expression
- target
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2320/00—Applications; Uses
- C12N2320/10—Applications; Uses in screening processes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2330/00—Production
- C12N2330/10—Production naturally occurring
Definitions
- the invention relates in general to microRNA molecules as well as various nucleic acid molecules relating thereto or derived therefrom.
- MicroRNAs are short RNA oligonucleotides of approximately 22 nucleotides that are involved in gene regulation. MicroRNAs regulate gene expression by targeting mRNAs for cleavage or translational repression. Although miRNAs are present in a wide range of species including C. elegans, Drosophilla and humans, they have only recently been identified. More importantly, the role of miRNAs in the development and progression of disease has only recently become appreciated. Deregulated miRNA expression is implicated in onset and progression of different diseases including, but not limited to embryonic malformations and cancers.
- miRNAs have been difficult to identify using standard methodologies.
- a limited number of miRNAs have been identified by extracting large quantities of RNA.
- MiRNAs have also been identified that contribute to the presentation of visibly discernable phenotypes.
- Expression array data shows that miRNAs are expressed in different developmental stages or in different tissues. The restriction of miRNAs to certain tissues or at limited developmental stages indicates that the miRNAs identified to date are likely only a small fraction of the total miRNAs.
- Computational approaches have recently been developed to identify the remainder of miRNAs in the genome. Tools such as MiRscan and MiRseeker have identified miRNAs that were later experimentally confirmed.
- the human genome contains 200-255 miRNA genes. These estimates are based on an assumption, however, that the miRNAs remaining to be identified will have the same properties as those miRNAs already identified. Based on the fundamental importance of miRNAs in mammalian biology and disease, the art needs to identify unknown miRNAs. The present invention satisfies this need and provides a significant number of miRNAs and uses therefore. [0006] Moreover, because of their potential broad use in treating and diagnosing different diseases, there is a need in the art (yet unmet) to develop methods of identification, isolation and also quatitation of miRNAs. The present invention addresses the need by disclosing efficient and sensitive methods and compositions for isolating and quantitating miRNAs from different samples, including those wherein there is only minimum amount of a starting material available.
- the nucleic acid may comprise a sequence of any of SEQ ID NOS: 1-42840282, the complement thereof, or a sequence at least 81% identical to 21 contiguous nucleotides thereof.
- the nucleic acid may also comprise the sequence of any of SEQ ID NOS: 3A-7A, the complement thereof, or a sequence at least 63% identical to 81 contiguous nucleotides thereof.
- the nucleic acid may also comprise the sequence of SEQ ID NOS: IA or 2 A, the complement thereof, or a sequence at least 63% identical to 81 contiguous nucleotides thereof.
- the nucleic acid may be from about 51 to about 250 nucleotides in length.
- the nucleic acid many comprise a modified base.
- a probe comprising the nucleic acid is also provided.
- a composition comprising the probe is also provided.
- a biochip comprising the probe is also provided.
- a method for detecting a disease-associated nucleic acid is also provided.
- a biological sample may be provided from which the level of a nucleic acid may be measured.
- the nucleic acid may comprise a sequence of any of SEQ ID NOS: 1-42840282 or 3A-7A.
- the nucleic acid may also comprise a sequence at least about 81% identical to about 21 contiguous nucleotides of any of SEQ ID NOS: 1-42840282 or3A-7A.
- a level of the nucleic acid higher than that of a control may be indicative of a disease.
- a method for identifying compound that modulates expression of a disease-associated rm ' RNA is also provided.
- a cell is provided that is capable of expressing a nucleic acid comprising a sequence of any of SEQ ID NOS: 1-26518 or 3A-7A.
- a cell may also be provided that is capable of expressing a nucleic acid comprising a sequence at least about 81% identical to about 21 contiguous nucleotides of any of SEQ ID NOS : 1 -26518 or 3 A-7A.
- the cell may be contacted with a candidate modulator.
- the level of expression of the nucleic acid may then be measured.
- a difference in the level of the nucleic acid compared to a control identifies the compound as a modulator of expression of the miRNA.
- a method of inhibiting expression of a target gene in a cell is also provided.
- a nucleic acid may be introduced into the cell in an amount sufficient to inhibit expression of the target gene.
- the target gene may comprise a binding site substantially identical to a binding site referred to in Table 5 or any of SEQ ID NOS: 26519-42840282.
- the nucleic acid may comprise a sequence of any of SEQ E) NOS: 3A-7A, SEQ ID NOS: 1-26518 or a variant thereof.
- the nucleic acid may also comprise a sequence at least about 81% identical to about 21 contiguous nucleotides of any of SEQ E) NOS : 3 A-7 A, SEQ E) NOS : 1 -26518 or a variant thereof.
- Expression of the target gene may be inhibited in vitro or in vivo.
- a method of increasing expression of a target gene in a cell is also provided.
- a nucleic acid may be introduced into the cell in an amount sufficient to increase expression of the target gene.
- the target gene may comprise a binding site substantially identical to a binding site referred to in Table 5 or any of SEQ E) NOS: 26519-42840282.
- the nucleic acid may comprise a sequence substantially complementary to any SEQ E) NOS: 3 A-7 A, SEQ E) NOS: 1-26518 or a variant thereof.
- the nucleic acid may also comprise a sequence substantially complementary to a sequence at least about 81% identical to about 21 contiguous nucleotides of any of SEQ E) NOS: 3 A-7 A, SEQ E) NOS: 1-26518 or a variant thereof. Expression of the target gene may be increased in vitro or in vivo.
- a method of treating a patient is also provided.
- the patient may suffer from a disorder set forth in Table 8.
- the patient may be administered a composition comprising a nucleic acid.
- the nucleic acid may comprise a sequence of any of SEQ E) NOS: 3A-7A, SEQ E) NOS: 1-26518, the complement thereof, or a sequence at least 81% identical to 21 contiguous nucleotides thereof.
- the nucleic acid may also comprise the sequence of SEQ E ) NOS: IA or 2A, any of SEQ E) NOS: 1-26518, the complement thereof, or a sequence at least 63% identical to 81 contiguous nucleotides thereof.
- the nucleic acid may be from about 51 to about 250 nucleotides in length.
- the nucleic acid many comprise a modified base.
- a method of detecting a target nucleic acid is provided.
- the targeted nucleic acid may be any nucleic acid, such as a miRNA.
- a nucleic acid comprising a short RNA sequence and a DNA sequence including a T7 RNA promoter sequence may be ligated to the miRNA molecule.
- Another oligonucleotide may be annealed to the T7 promoter region to enable RNA polymerize binding to the double strand. Repeated cycles of initiation and product release may then be performed. As a result a linear amplification of the transcript that includes a complimentary sequence to the original natural miRNA is achieved.
- nucleotides that are labeled may be incorporated to the transcript, which may be useful in the later detection of the transcript by a variety of assays.
- this method may provide detection of such target nucleic acids in methods such as Luminex.
- LNA locked nucleic acid
- the compact discs contain the following: SEQ_01.txt (665,724 KB, 2/7/2006), SEQ_02.txt (673,696 KB, 2/6/2006), SEQ_03.txt (672,326 KB, 2/6/2006), SEQ_04.txt (673,986 KB 5 2/6/2006), SEQ_05.txt (668,866 KB, 2/6/2006), SEQ_06.txt (666,389 KB, 2/6/2006), SEQ_07.txt (673,699 KB, 2/6/2006), SEQ_08.txt (668,601 KB, 2/6/2006), SEQ_09.txt (667,636 KB, 2/6/2006), SEQ_10.txt (669,467 KB, 2/6/2006), SEQ_ll.txt (668,877 KB, 2/6/2006), SEQ_12.t
- Figure 1 demonstrates a model of maturation for miRNAs.
- FIG. 2 shows a schematic illustration of the MC19 cluster on 19ql3.42.
- Panel A shows the ⁇ 500,000bp region of chromosome 19, from 58,580,001 to 59,080,000 (according to the May 2004 USCS assembly), in which the cluster is located including the neighboring protein-coding genes.
- the MC19-1 cluster is indicated by a rectangle. Mir-371, mir-372, and mir-373 are indicted by lines. Protein coding genes flanking the cluster are represented by large arrow-heads.
- Panel B shows a detailed structure of the MC19-1 miRNA cluster. A region of ⁇ 102,000bp, from 58,860,001 to 58,962,000 (according to the May 2004 USCS assembly), is presented.
- MiRNA precursors are represented by a black bars. It should be noted that all miRNAs are at the same orientation from left to right. Shaded areas around miRNA precursors represent repeating units in which the precursor is embedded. The location of mir-371, mir-372, and mir-373, is also presented.
- Figure 3 is a graphical representation of multiple sequence alignment of 35 human repeat units at distinct size of ⁇ 690nt (A) and 26 chimpanzees repeat units (B).
- the graph was generated by calculating a similarity score for each position in the alignment with an averaging sliding window of IOnt (Maximum score -1, minimum score-0).
- the repeat unit sequences were aligned by ClustalW program. Each position of the resulting alignment was assigned a score which represented the degree of similarity at this position.
- the region containing the miRNA precursors is bordered by vertical lines.
- the exact location of the mature miRNAs derived from the 5' stems (5p) and 3 ' stems (3p) of the precursors is indicted by vertical lines.
- Figure 4 shows sequence alignments of the 43 A-type pre-miRNAs of the MC 19-1 cluster.
- Panel A shows the multiple sequence alignment with the Position of the mature miRNAs marked by a frame. The consensus sequence is shown at the bottom. conserveed nucleotides are colored as follows: black-100%, dark grey- 80% to 99%, and clear grey- 60% to 79%.
- Panel B shows alignments of consensus mature A-type miRNAs with the upstream human cluster of mir- 371, mir-372, miR-373.
- Panel C shows alignments of consensus mature A-type miRNAs with the hsa-mir-371-373 mouse orthologous cluster.
- Figure 5 shows expression analysis of the MC19-1 miRNAs.
- Panel A shows a Northern blot analysis of two selected A-type miRNAs. Expression was analyzed using total RNA from human brain (B), liver (L), thymus (T), placenta (P) and HeLa cells (H). The expression of mir-98 and ethidium bromide staining of the tRNA band served as control.
- Panel B shows RT-
- PCR analysis of the mRNA transcript containing the A-type miRNA precursors Reverse transcription of 5yg total RNA from placenta was performed using oligo-dT. This was followed by PCR using the denoted primers (indicated by horizontal arrows). The region examined is illustrated at the top. Vertical black bars represent the pre-miRNA; shaded areas around the pre- miRNAs represent the repeating units; the location of four ESTs is indicted at the right side; the poly-A site, as found in the ESTs and located downstream to a AATAAA consensus, is indicated by a vertical arrow. The fragments expected from RT-PCR using three primer combinations are indicated below the illustration of the cluster region. The results of the RT-PCR analysis are presented below the expected fragments. Panel C shows the sequencing strategy of the FR2 fragment. The fragment was cloned into the pTZ57R ⁇ T vector and sequenced using external and internal primers.
- Figure 6 shows the predicted hairpin structure of a miRNA precursor (SEQ ID NO: 2A) encoded by the 13_12 gene.
- the residues marked with underlines are predicted to be the mature miRNA (3 1 ) and miRNA* (5').
- Figure 7 shows an alignment of predicted and/or validated miRNA precursors homolous to 13_12 which encode the following miRNAs: mmu-mir-193 (SEQ ID NO: 8A), rno-mir-193
- Figure 8 shows an alignment of the following 13_12-derived miRNAs that were cloned:
- 19061 (SEQ ID NO: 3A), 19062 (SEQ ID NO: 4A), 19063 (SEQ ID NO: 5A), (SEQ ID NO: 6A) and 19065 (SEQ ID NO: 7A).
- Figure 9 shows a Northern blot analysis of 13_12 expression in brain (B), liver (L), thymus (T), placenta (P) and HeIa cells (H) using 40 ⁇ g total RNA. Expression of the has-mir-
- has-mir-98 was used as a control. Equal loading of the gel before transfer to membrane was monitored by ethidium bromide staining of the tRNA band (bottom). The expression of has-mir-98 was examined for reference.
- Figure 10 shows the relative expression of 13 12 and other miRNA genes in prostate and lung tumors, with the Y-axis being shown in log base 2.
- Figure 11 shows that inhibition of 13_12 caused a decrease in the proliferation of PC-3 cells.
- Figure 12 shows the comparative inhibition of the 13_12 inhibitor and other miRNA inhibitors in PC-3 cells.
- Figure 13 shows the comparative inhibition of the 13 12 inhibitor and other miRNA inhibitors in A549 cells.
- Figure 13 shows linear amplification of microKNA.
- An adaptor containing a DNA sequence complementary to an RNA promoter is ligated to a microRNA.
- a primer containing the DNA sequence for the promoter is then annealed to the miRNA-adaptor hybrid. Labeled reverse complements to the miRNA are then generated by RNA polymerase.
- Figure 14 shows sequences for an adaptor (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: IB), annealing primer (SEQ ID NO: I
- Nucleic acids are provided related to miRNAs, precursors thereto, and targets thereof. Such nucleic acids may be useful for diagnostic and prognostic purposes, and also for modifying target gene expression. Also provided are methods and compositions that may be useful, among other things, for diagnostic and prognostic purposes. Other aspects of the invention will become apparent to the skilled artisan by the following description of the invention. 1. Definitions
- Animal as used herein may mean fish, amphibians, reptiles, birds, and mammals, such as mice, rats, rabbits, goats, cats, dogs, cows, apes and humans.
- Attached or “immobilized” as used herein to refer to a probe and a solid support may mean that the binding between the probe and the solid support is sufficient to be stable under conditions of binding, washing, analysis, and removal.
- the binding may be covalent or non- covalent. Covalent bonds may be formed directly between the probe and the solid support or may be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules.
- Non-covalent binding may be one or more of electrostatic, hydrophilic, and hydrophobic interactions.
- non-covalent binding is the covalent attachment of a molecule, such as streptavidin, to the support and the non-covalent binding of a biotinylated probe to the streptavidin. Immobilization may also involve a combination of covalent and non-covalent interactions. c. biological sample
- Biological sample as used herein may mean a sample of biological tissue or fluid that comprises nucleic acids. Such samples include, but are not limited to, tissue or fluid isolated from animals. Biological samples may also include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood, plasma, serum, sputum, stool, tears, mucus, hair, and skin. Biological samples also include explants and primary and/or transformed cell cultures derived from animal or patient tissues. A biological sample may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods described herein in vivo. Archival tissues, such as those having treatment or outcome history, may also be used. d. complement
- “Complement” or “complementary” as used herein to refer to a nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen base pairing between nucleotides or nucleotide analogs of nucleic acid molecules. e. differential expression
- differential expression may mean qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue.
- a differentially expressed gene may qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus disease tissue. Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more states.
- a qualitatively regulated gene may exhibit an expression pattern within a state or cell type which may be detectable by standard techniques. Some genes may be expressed in one state or cell type, but not in both.
- the difference in expression may be quantitative, e.g., in that expression is modulated, either up-regulated, resulting in an increased amount of transcript, or down-regulated, resulting in a decreased amount of transcript.
- the degree to which expression differs need only be large enough to quantify via standard characterization techniques such as expression arrays, quantitative reverse transcriptase PCR, northern analysis, and RNase protection. f. gene
- Gene used herein may be a natural (e.g., genomic) or synthetic gene comprising transcriptional and/or translational regulatory sequences and/or a coding region and/or non- translated sequences (e.g., introns, 5'- and 3 '-untranslated sequences).
- the coding region of a gene may be a nucleotide sequence coding for an amino acid sequence or a functional RNA, such as tRNA, rRNA, catalytic RNA, siRNA, miRNA or antisense RNA.
- a gene may also be an mRNA or cDNA corresponding to the coding regions (e.g., exons and miRNA) optionally comprising 5'- or 3 '-untranslated sequences linked thereto.
- a gene may also be an amplified nucleic acid molecule produced in vitro comprising all or a part of the coding region and/or 5'- or 3 '-untranslated sequences linked thereto.
- Host cell used herein may be a naturally occurring cell or a transformed cell that may contain a vector and may support replication of the vector.
- Host cells may be cultured cells, explants, cells in vivo, and the like.
- Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells, such as CHO and HeLa. h. identity
- Identity as used herein in the context of two or more nucleic acids or polypeptide sequences, may mean that the sequences have a specified percentage of residues that are the same over a specified region. The percentage may be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity.
- Label as used herein may mean a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
- useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and other entities which can be made detectable.
- a label may be incorporated into nucleic acids and proteins at any position. j. nucleic acid
- Nucleic acid or "oligonucleotide” or “polynucleotide” used herein may mean at least two nucleotides covalently linked together.
- the depiction of a single strand also defines the sequence of the complementary strand.
- a nucleic acid also encompasses the complementary strand of a depicted single strand.
- Many variants of a nucleic acid may be used for the same purpose as a given nucleic acid.
- a nucleic acid also encompasses substantially identical nucleic acids and complements thereof.
- a single strand provides a probe that may hybridize to a target sequence under stringent hybridization conditions.
- nucleic acids may be single stranded or double stranded, or may contain portions of both double stranded and single stranded sequence.
- the nucleic acid may be DNA, both genomic and cDNA, RNA, or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine and isoguanine.
- Nucleic acids may be obtained by chemical synthesis methods or by recombinant methods.
- a nucleic acid will generally contain phosphodiester bonds, although nucleic acid analogs may be included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages and peptide nucleic acid backbones and linkages.
- Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, which are incorporated by reference.
- Nucleic acids containing one or more non-naturally occurring or modified nucleotides are also included within one definition of nucleic acids.
- the modified nucleotide analog may be located for example at the 5'-end and/or the 3'-end of the nucleic acid molecule.
- Representative examples of nucleotide analogs may be selected from sugar- or backbone-modified ribonucleotides. It should be noted, however, that also nucleobase-modified ribonucleotides, i.e. ribonucleotides, containing a non- naturally occurring nucleobase instead of a naturally occurring nucleobase such as uridines or cytidines modified at the 5-position, e.g.
- the T- OH-group may be replaced by a group selected from H, OR, R, halo, SH, SR, NH 2 , NHR, NR 2 or CN, wherein R is C 1 -C 6 alkyl, alkenyl or alkynyl and halo is F, Cl, Br or I.
- Modified nucleotides also include nucleotides conjugated with cholesterol through, e.g., a hydroxyprolinol linkage as described in Rrutzfeldt et al., Nature (Oct. 30, 2005), Soutschek et al., Nature 432:173-178 (2004), and U.S. Patent Publication No. 20050107325, which are incorporated herein by reference.
- operably linked used herein may mean that expression of a gene is under the control of a promoter with which it is spatially connected.
- a promoter may be positioned 5' (upstream) or 3' (downstream) of a gene under its control.
- the distance between the promoter and a gene may be approximately the same as the distance between that promoter and the gene it controls in the gene from which the promoter is derived. As is known in the art, variation in this distance may be accommodated without loss of promoter function.
- Probe as used herein may mean an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. Probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. There may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids described herein. However, if the number of mutations is so great that no hybridization can occur under even the least stringent of hybridization conditions, the sequence is not a complementary target sequence.
- a probe may be single stranded or partially single and partially double stranded. The strandedness of the probe is dictated by the structure, composition, and properties of the target sequence. Probes may be directly labeled or indirectly labeled such as with biotin to which a streptavidin complex may later bind. m. promoter
- Promoter may mean a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell.
- a promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
- a promoter may also comprise distal enhancer or repressor elements, which can be located as much as . several thousand base pairs from the start site of transcription.
- a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
- a promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
- promoters include the bacteriophage T7 promoter, bacteriophage T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, SV40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV IE promoter, SV40 early promoter or S V40 late promoter and the CMV IE promoter.
- Selectable marker used herein may mean any gene which confers a phenotype on a host cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct.
- selectable markers include the ampicillin-resistance gene (Amp 1 ), tetracycline-resistance gene (Tc r ), bacterial kanamycin-resistance gene (Kan 1 ), zeocin resistance gene, the AURI-C gene which confers resistance to the antibiotic aureobasidin A, phosphinothricin-resistance gene, neomycin phosphotransferase gene (nptll), hygromycin-resistance gene, beta-glucuronidase (GUS) gene, chloramphenicol acetyltransferase (CAT) gene, green fluorescent protein (GFP)-encoding gene and luciferase gene.
- Amp 1 ampicillin-resistance gene
- Stringent hybridization conditions used herein may mean conditions under which a first nucleic acid sequence (e.g., probe) will hybridize to a second nucleic acid sequence (e.g., target), such as in a complex mixture of nucleic acids. Stringent conditions are sequence-dependent and will be different in different circumstances. Stringent conditions may be selected to be about 5-10 0 C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength pH.
- the T m may be the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
- Stringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., about 10- 50 nucleotides) and at least about 60°C for long probes (e.g., greater than about 50 nucleotides).
- Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
- destabilizing agents such as formamide.
- a positive signal maybe at least 2 to 10 times background hybridization.
- Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65 0 C, with wash in 0.2x SSC, and 0.1% SDS at 65°C. p. substantially complementary
- substantially complementary used herein may mean that a first sequence is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the complement of a second sequence over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides, or that the two sequences hybridize under stringent hybridization conditions. q. substantially identical
- substantially identical used herein may mean that a first and second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with respect to nucleic acids, if the first sequence is substantially complementary to the complement of the second sequence. r. target
- Target as used herein may mean a polynucleotide that may be bound by one or more probes under stringent hybridization conditions. s. terminator
- Terminator used herein may mean a sequence at the end of a transcriptional unit which signals termination of transcription.
- a terminator may be a 3 '-non-translated DNA sequence containing a polyadenylation signal, which may facilitate the addition of polyadenylate sequences to the 3 '-end of a primary transcript.
- a terminator may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
- terminators include the SV40 polyadenylation signal, HSV TK polyadenylation signal, CYCl terminator, ADH terminator, SPA terminator, nopaline synthase (NOS) gene terminator of Agrobacterium tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the Rubisco small subunit gene (SSU) gene terminator sequences, subclover stunt virus (SCSV) gene sequence terminators, rho-independent E. coli terminators, and the lacZ alpha terminator.
- SSU Rubisco small subunit gene
- SCSV subclover stunt virus
- Variant used herein to refer to a nucleic acid may mean (i) a portion of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a nucleic acid that is substantially identical to a referenced nucleic acid or the complement thereof; or (iv) a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
- vectors a nucleic acid that hybridizes under stringent conditions to the referenced nucleic acid, complement thereof, or a sequences substantially identical thereto.
- Vector used herein may mean a nucleic acid sequence containing an origin of replication.
- a vector may be a plasmid, bacteriophage, bacterial artificial chromosome or yeast artificial chromosome.
- a vector may be a DNA or RNA vector.
- a vector may be either a self- replicating extrachromosomal vector or a vector which integrates into a host genome. 2.
- a gene coding for a miRNA may be transcribed leading to production of an miRNA precursor known as the pri-miRNA.
- the pri-miRNA may be part of a polycistronic RNA comprising multiple pri-miRNAs.
- the pri-miRNA may form a hairpin with a stem and loop. As indicated on Figure 1, the stem may comprise mismatched bases.
- the hairpin structure of the pri-miRNA may be recognized by Drosha, which is an RNase III endonuclease.
- Drosha may recognize terminal loops in the pri-miRNA and cleave approximately two helical turns into the stem to produce a 60-70 nt precursor known as the pre- miRNA.
- Drosha may cleave the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5' phosphate and ⁇ 2 nucleotide 3' overhang.
- Approximately one helical turn of stem ( ⁇ 10 nucleotides) extending beyond the Drosha cleavage site may be essential for efficient processing.
- the pre-miRNA may then be actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Ex- portin-5.
- the pre-miRNA may be recognized by Dicer, which is also an RNase III endonuclease. Dicer may recognize the double-stranded stem of the pre-miRNA. Dicer may also recognize the 5' phosphate and 3' overhang at the base of the stem loop. Dicer may cleave off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5' phosphate and ⁇ 2 nucleotide 3' overhang. The resulting siRNA-like duplex, which may comprise mismatches, comprises the mature miRNA and a similar-sized fragment known as the miRNA*. The miRNA and miRNA* maybe derived from opposing arms of the pri-miRNA and pre- miRNA. MiRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
- RISC RNA-induced silencing complex
- the miRNA strand of the miRNA:miRNA* duplex When the miRNA strand of the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* may be removed and degraded.
- the strand of the miRNA:miRNA* duplex that is loaded into the RISC may be the strand whose 5' end is less tightly paired. In cases where both ends of the miRNA:miRNA* have roughly equivalent 5' pairing, both miRNA and miRNA* may have gene silencing activity.
- the RISC may identify target nucleic acids based on high levels of complementarity between the miRNA and the mRNA, especially by nucleotides 2-8 of the miRNA. Only one case has been reported in animals where the interaction between the miRNA and its target was along the entire length of the miRNA. This was shown for mir-196 and Hox B8 and it was further shown that mir-196 mediates the cleavage of the Hox B8 mRNA (Yekta et al 2004, Science 304-594). Otherwise, such interactions are known only in plants (Bartel & Bartel 2003, Plant Physiol 132-709).
- the target sites in the mRNA may be in the 5' UTR, the 3' UTR or in the coding region.
- multiple miRNAs may regulate the same mRNA target by recognizing the same or multiple sites.
- the presence of multiple miRNA binding sites in most genetically identified targets may indicate that the cooperative action of multiple RISCs provides the most efficient translational inhibition.
- MiRNAs may direct the RISC to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression.
- the miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut may be between the nucleotides pairing to residues 10 and 11 of the miRNA.
- the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
- nucleic acids are provided herein.
- the nucleic acid may comprise the sequence of SEQ ID NOS: 1- ⁇ 42840282 or variants thereof.
- the variant may be a complement of the referenced nucleotide sequence.
- the variant may also be a nucleotide sequence that is substantially identical to the referenced nucleotide sequence or the complement thereof.
- the variant may also be a nucleotide sequence which hybridizes under stringent conditions to the referenced nucleotide sequence, complements thereof, or nucleotide sequences substantially identical thereto.
- the nucleic acid may have a length of from 10 to 250 nucleotides.
- the nucleic acid may have a length of at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or 250 nucleotides.
- the nucleic acid may be synthesized or expressed in a cell (in vitro or in vivo) using a synthetic gene described herein.
- the nucleic acid may be synthesized as a single strand molecule and hybridized to a substantially complementary nucleic acid to form a duplex.
- the nucleic acid may be introduced to a cell, tissue or organ in a single- or double-stranded form or capable of being expressed by a synthetic gene using methods well known to those skilled in the art, including as described in U.S. Patent No. 6,506,559 which is incorporated by reference. a. Pri-miRNA
- the nucleic acid may comprise a sequence of a pri-miRNA or a variant thereof.
- the pri- miRNA sequence may comprise from 45-250, 55-200, 70-150 or 80-100 nucleotides.
- the sequence of the pri-miRNA may comprise a pre-miRNA, miRNA and miRNA*, as set forth herein, and variants thereof.
- the sequence of the pri-miRNA may comprise the sequence of SEQ ID NOS: 1-8857-26518 or variants thereof.
- the pri-miRNA may form a hairpin structure.
- the hairpin may comprise a first and second nucleic acid sequence that are substantially complimentary.
- the first and second nucleic acid sequence may be from 37-50 nucleotides.
- the first and second nucleic acid sequence may be separated by a third sequence of from 8-12 nucleotides.
- the hairpin structure may have a free energy less than -25 Kcal/mole as calculated by the Vienna algorithm with default parameters, as described in Hofacker et al., Monatshefte f. Chemie 125: 167-188 (1994), the contents of which are incorporated herein.
- the hairpin may comprise a terminal loop of 4-20, 8-12 or 10 nucleotides.
- the pri-miRNA may comprise at least 19% adenosine nucleotides, at least 16% cytosine nucleotides, at least 23% thymine nucleotides and at least 19% guanine nucleotides.
- the nucleic acid may also comprise a sequence of a pre-miRNA or a variant thereof.
- the pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
- the sequence of the pre-miRNA may comprise a miRNA and a miRNA* as set forth herein,
- the sequence of the pre-miRNA may also be that of a pri-miRNA excluding from 0-160 nucleotides from the 5' and 3' ends of the pri-miRNA.
- the sequence of the pre-miRNA may comprise the sequence of SEQ ID NOS: 1-8857-26518 or variants thereof.
- the nucleic acid may also comprise a sequence of a miRNA (including miRNA*) or a variant thereof.
- the miRNA sequence may comprise from 13-33, 18-24 or 21-23 nucleotides.
- the miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
- the sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA.
- the sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
- the sequence of the miRNA may comprise the sequence of SEQ ID NOS: 8857-26518 or variants thereof.
- the nucleic acid may also comprise a sequence of an anti-miRNA that is capable of blocking the activity of a miRNA or miRNA*, such as by binding to the pri-miRNA, pre- miRNA, miRNA or miRNA* (e.g. antisense or RNA silencing), or by binding to the target binding site.
- the anti-miRNA may comprise a total of 5-100 or 10-60 nucleotides.
- the anti- miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
- the sequence of the anti-rm ' RNA may comprise (a) at least 5 nucleotides that are substantially identical or complimentary to the 5' of a miRNA and at least 5-12 nucleotides that are substantially complimentary to the flanking regions of the target site from the 5' end of the miRNA, or (b) at least 5-12 nucleotides that are substantially identical or complimentary to the 3' of a miRNA and at least 5 nucleotide that are substantially complimentary to the flanking region of the target site from the 3' end of the miRNA.
- the sequence of the anti-miRNA may comprise the compliment of SEQ E) NOS: 8857-26518 or variants thereof.
- the nucleic acid may also comprise a sequence of a target miRNA binding site, or a variant thereof.
- the target site sequence may comprise a total of 5-100 or 10-60 nucleotides.
- the target site sequence may also comprise a total ofat least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 or 63 nucleotides.
- the target site sequence may comprise at least 5 nucleotides of the sequence of SEQ ID NOS: 8857-26518 or a target gene binding site referred to in Table 5.
- a synthetic gene comprising a nucleic acid described herein operably linked to a transcriptional and/or translational regulatory sequence.
- the synthetic gene may be capable of modifying the expression of a target gene with a binding site for a nucleic acid described herein. Expression of the target gene may be modified in a cell, tissue or organ.
- the synthetic gene may be synthesized or derived from naturally-occurring genes by standard recombinant techniques.
- the synthetic gene may also comprise terminators at the 3'-end of the transcriptional unit of the synthetic gene sequence.
- the synthetic gene may also comprise a selectable marker.
- a vector comprising a synthetic gene described herein.
- the vector may be an expression vector.
- An expression vector may comprise additional elements.
- the expression vector may have two replication systems allowing it to be maintained in two organisms, e.g., in one host cell for expression and in a second host cell (e.g., bacteria) for cloning and amplification.
- the expression vector may contain at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
- the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector.
- the vector may also comprise a selectable marker gene to allow the selection of transformed host cells.
- a host cell comprising a vector, synthetic gene or nucleic acid described herein.
- the cell may be a bacterial, fungal, plant, insect or animal cell.
- a probe is also provided comprising a nucleic acid described herein. Probes may be used for screening and diagnostic methods, as outlined below. The probe may be attached or immobilized to a solid substrate, such as a biochip.
- the probe may have a length of from 8 to 500, 10 to 100 or 20 to 60 nucleotides.
- the probe may also have a length of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280 or 300 nucleotides.
- the probe may further comprise a linker sequence of from 10-60 nucleotides.
- a biochip is also provided.
- the biochip may comprise a solid substrate comprising an attached probe or plurality of probes described herein.
- the probes may be capable of hybridizing to a target sequence under stringent hybridization conditions.
- the probes may be attached at spatially defined address on the substrate. More than one probe per target sequence may be used, with either overlapping probes or probes to different sections of a particular target sequence.
- the probes may be capable of hybridizing to target sequences associated with a single disorder.
- the probes may be attached to the biochip in a wide variety of ways, as will be appreciated by those in the art.
- the probes may either be synthesized first, with subsequent attachment to the biochip, or may be directly synthesized on the biochip.
- the solid substrate may be a material that may be modified to contain discrete individual sites appropriate for the attachment or association of the probes and is amenable to at least one J
- substrates include glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses and plastics.
- the substrates may allow optical detection without appreciably fluorescing.
- the substrate may be planar, although other configurations of substrates may be used as well.
- probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
- the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
- the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
- the biochip may be derivatized with a chemical functional group including, but not limited to, amino groups, carboxyl groups, oxo groups or thiol groups. Using these functional groups, the probes may be attached using functional groups on the probes either directly or indirectly using a linkers.
- the probes may be attached to the solid support by either the 5' terminus, 3' terminus, or via an internal nucleotide.
- the probe may also be attached to the solid support non-covalently.
- biotinylated oligonucleotides can be made, which may bind to surfaces covalently coated with streptavidin, resulting in attachment.
- probes may be synthesized on the surface using techniques such as photopolymerization and photolithography.
- a method of identifying a nucleic acid associated with a disease or a pathological condition comprises measuring a level of the nucleic acid in a sample that is different than the level of a control. Detection may be performed by contacting the sample with a probe or biochip described herein and detecting the amount of hybridization. PCR may be used to amplify nucleic acids in the sample, which may provide higher sensitivity. [0087] The level of the nucleic acid in the sample may also be compared to a control cell (e.g., a normal cell) to determine whether the nucleic acid is differentially expressed (e.g., overexpressed or underexpressed).
- a control cell e.g., a normal cell
- the ability to identify miRNAs that are differentially expressed in pathological cells compared to a control can provide high-resolution, high-sensitivity datasets which may be used in the areas of diagnostics, prognostics, therapeutics, drug development, pharmacogenetics, biosensor development, and other related areas.
- An expression profile generated by the current methods may be a "fingerprint" of the state of the sample with respect to a number of miRNAs. While two states may have any particular miRNA similarly expressed, the evaluation of a number of miRNAs simultaneously allows the generation of a gene expression profile that is characteristic of the state of the cell. That is, normal tissue may be distinguished from diseased tissue. By comparing expression profiles of tissue in known different disease states, information regarding which miRNAs are associated in each of these states may be obtained. Then, diagnosis may be performed or confirmed to determine whether a tissue sample has the expression profile of normal or disease tissue. This may provide for molecular diagnosis of related conditions.
- the expression level of a disease-associated nucleic acid is information in a number of ways. For example, a differential expression of a disease-associated nucleic acid compared to a control may be used as a diagnostic that a patient suffers from the disease. Expression levels of a disease-associated nucleic acid may also be used to monitor the treatment and disease state of a patient. Furthermore, expression levels of a disease-associated miRNA may allow the screening of drug candidates for altering a particular expression profile or suppressing an expression profile associated with disease.
- a target nucleic acid may be detected and levels of the target nucleic acid measured by contacting a sample comprising the target nucleic acid with a biochip comprising an attached probe sufficiently complementary to the target nucleic acid and detecting hybridization to the probe above control levels.
- the target nucleic acid may also be detected by immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing a labelled probe with the sample. Similarly, the target nucleic may also be detected by immobilizing the labeled probe to a solid support and hybridizing a sample comprising a labeled target nucleic acid. Following washing to remove the non-specific hybridization, the label may be detected. [0091] The target nucleic acid may also be detected in situ by contacting permeabilized cells or tissue samples with a labeled probe to allow hybridization with the target nucleic acid. Following washing to remove the non-specifically bound probe, the label may be detected.
- These assays can be direct hybridization assays or can comprise sandwich assays, which include the use of multiple probes, as is generally outlined in U.S. Pat. Nos. 5,681,702; 5,597,909; 5,545,730; 5,594,117; 5,591,584; 5,571,670; 5,580,731; 5,571,670; 5,591,584; 5,624,802; 5,635,352; 5,594,118; 5,359,100; 5,124,246; and 5,681,697, each of which is hereby incorporated by reference.
- hybridization conditions may be used, including high, moderate and low stringency conditions as outlined above.
- the assays may be performed under stringency conditions which allow hybridization of the probe only to the target.
- Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, or organic solvent concentration.
- Hybridization reactions may be accomplished in a variety of ways. Components of the reaction may be added simultaneously, or sequentially, in different orders.
- Reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors and antimicrobial agents may also be used as appropriate, depending on the sample preparation methods
- a method of diagnosis comprises detecting a differential expression level of a disease-associated nucleic acid in a biological sample.
- the sample may be derived from a patient. Diagnosis of a disease state in a patient may allow for prognosis and selection of therapeutic strategy. Further, the developmental stage of cells may be classified by determining temporarily expressed disease-associated nucleic acids.
- In situ hybridization of labeled probes to tissue arrays may be performed. When comparing the fingerprints between an individual and a standard, the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes.
- a method of screening therapeutics comprises contacting a pathological cell capable of expressing a disease related nucleic acid with a candidate therapeutic and evaluating the effect of a drug candidate on the expression profile of the disease associated nucleic acid. Having identified the differentially expressed nucleic acid, a variety of assays may be executed. Test compounds may be screened for the ability to modulate gene expression of the disease associated nucleic acid. Modulation includes both an increase and a decrease in gene expression.
- the test compound or drug candidate may be any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the disease phenotype or the expression of the disease associated nucleic acid.
- Drug candidates encompass numerous chemical classes, such as small organic molecules having a molecular weight of more than 100 and less than about 500, 1,000, 1,500, 2,000 or 2,500 daltons.
- Candidate compounds may comprise functional groups necessary. for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
- the candidate agents may comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
- Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
- Combinatorial libraries of potential modulators may be screened for the ability to bind to the disease associated nucleic acid or to modulate the activity thereof.
- the combinatorial library may be a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical building blocks such as reagents. Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art.
- Such combinatorial chemical libraries include, but are not limited to, peptide libraries encoded peptides, benzodiazepines, diversomers such as hydantoins, benzodiazepines and dipeptide, vinylogous polypeptides, analogous organic syntheses of small compound libraries, oligocarbamates, and/or peptidyl phosphonates, nucleic acid libraries, peptide nucleic acid libraries, antibody libraries, carbohydrate libraries, and small organic molecule libraries. 10.
- a method of reducing expression of a target gene in a cell, tissue or organ is also provided.
- Expression of the target gene may be reduced by expressing a nucleic acid described herein that comprises a sequence substantially complementary to one or more binding sites of the target mRNA.
- the nucleic acid may be a miRNA or a variant thereof.
- the nucleic acid may also be pri-miRNA, pre-miRNA, or a variant thereof, which may be processed to yield a miRNA.
- the expressed miRNA may hybridize to a substantially complementary binding site on the target mRNA, which may lead to activation of RISC-mediated gene silencing.
- the target of gene silencing may be a protein that causes the silencing of a second protein. By repressing expression of the target gene, expression of the second protein may be increased. Examples for efficient suppression of miRNA expression are the studies by Esau et al 2004 JBC 275-52361; and Cheng et al 2005 Nucleic Acids Res. 33-1290, which is incorporated herein by reference.
- a method of increasing expression of a target gene in a cell, tissue or organ is also provided.
- Expression of the target gene may be increased by expressing a nucleic acid described herein that comprises a sequence substantially complementary to a pri-miRNA, pre- miRNA, miRNA or a variant thereof.
- the nucleic acid may be an anti-miRNA.
- the anti- miRNA may hybridize with a pri-miRNA, pre-miRNA or miRNA, thereby reducing its gene repression activity.
- Expression of the target gene may also be increased by expressing a nucleic acid that is substantially complementary to a portion of the binding site in the target gene, such that binding of the nucleic acid to the binding site may prevent miRNA binding.
- a method of modulating a disease or disorder associated with developmental dysfunctions is also provided.
- the disease or disorder may be cancer, such as prostate or lung cancer.
- the nucleic acid molecules described herein may be used as a modulator of the expression of genes which are at least partially complementary to said nucleic acid.
- miRNA molecules may act as target for therapeutic screening procedures, e.g. inhibition or activation of miRNA molecules might modulate a cellular differentiation process, e.g. proliferation or apoptosis.
- miRNA molecules may be used as starting materials for the manufacture of sequence-modified miRNA molecules, in order to modify the target-specificity thereof, e.g. an oncogene, a multidrug-resistance gene or another therapeutic target gene. Further, miRNA molecules can be modified, in order that they are processed and then generated as double-stranded siRNAs which are again directed against therapeutically relevant targets. Furthermore, miRNA molecules may be used for tissue reprogramming procedures, e.g. a differentiated cell line might be transformed by expression of miRNA molecules into a different cell type or a stem cell.
- tissue reprogramming procedures e.g. a differentiated cell line might be transformed by expression of miRNA molecules into a different cell type or a stem cell.
- a pharmaceutical composition is also provided.
- the composition may comprise a nucleic acid described herein and optionally a pharmaceutically acceptable carrier.
- the compositions may be used for diagnostic or therapeutic applications.
- the pharmaceutical composition may be administered by known methods, including wherein a nucleic acid is introduced into a desired target cell in vitro or in vivo. Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation, microinjection, viral methods and cationic liposomes.
- kits comprising a nucleic acid described herein together with any or all of the following: assay reagents, buffers, probes and/or primers, and sterile saline or another pharmaceutically acceptable emulsion and suspension base.
- the kits may include instructional materials containing directions (e.g., protocols) for the practice of the methods described herein.
- a method of synthesizing the reverse-complement of a target nucleic acid is also provided.
- a first nucleic acid may be provided comprising the target nucleic acid and an adapter nucleic acid.
- the target nucleic acid may be 5' of the second nucleic acid.
- a second nucleic acid may then be provided.
- a portion of the second nucleic acid may be substantially complementary to a portion of the adapter nucleic acid.
- the second nucleic acid may be annealed to the adapter nucleic acid of the first nucleic acid to form an annealed complex.
- the second nucleic acid may then be extended from its 3' end, which may lead to the synthesis of the reverse-complement of the target nucleic acid. Extension may occur in a solution comprising labeled nucleotides, such as biotinylated UTP or/and CTP.
- the extended second nucleic acid may then be displaced from the first nucleic acid.
- the extended second nucleic acid may be displaced using heat denaturation.
- Another cycle of extension may be performed by providing a second nucleic acid, a portion of which may be substantially complementary to a portion of the adapter nucleic acid.
- the second nucleic acid may then be annealed to the adapter nucleic acid of the first nucleic acid to form an annealed complex.
- the second nucleic acid may then be extended from its 3' end, which may lead to the synthesis of another reverse-complement of the target nucleic acid.
- Additional cycles of displacement and extension may then be performed until a desirable amount of reverse- complement of the target nucleic acid has been produced.
- the number of cycles of displacement and extension performed may be at least 1, 10, 50, 100, 1000 or 10,000.
- the resulting reverse-complement transcripts may be utilitized for a number of different purposes.
- the amplified sequence may be cloned.
- the reverse- complement transcripts transcript may be used in a variety of different methods.
- the amplification of the transcript maybe linear, which may lead to improved quantitative analysis.
- the first nucleic acid may be formed by ligating the target nucleic acid to the adapter nucleic acid. Any ligase may be used to ligate the adaptor to the target nucleic acid to the adaptor nucleic acid, such as T4 RNA ligase.
- the ligated target nucleic acid- adaptor nucleic acid hybrid may be purified by a variety of methods including, but not limited to, acrylamide gel electrophoresis. The hybrid molecules may then be excised from the gel and extracted by any method, for example, by dialysis.
- the target nucleic acid may be derived from a biological sample comprising nucleic acids.
- the target nucleic acid may be enriched by size-fractionation.
- the target nucleic acid may any nucleic acid.
- the target nucleic acid may comprise RKA.
- the RNA may comprise 18 to 24 nucleotides.
- the RNA may be a miRNA or a shRNA.
- the adapter nucleic acid may comprise DNA.
- the adapter nucleic acid may comprise 10 to 20 nucleotides of DNA.
- the adapter nucleic acid may also comprise RNA.
- the adapter nucleic acid may comprise 1 to 5 nucleotides of RNA.
- the adapter nucleic acid may also comprise DNA and RNA.
- the adapter nucleic acid may comprise 10 to 20 nucleotides of DNA.
- the RNA of the adapter nucleic acid may be 5' of the DNA.
- the adapter nucleic acid may comprise a 5 '-phosphate.
- the adapter nucleic acid may also comprise a 3'-IdT.
- the second nucleic acid may be any nucleic acid.
- the second nucleic may comprise DNA. c. Polymerase
- the second nucleic acid may be extended using a suitable polymerase.
- suitable polymerase include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase and SP6 RNA polymerase.
- a double-stranded portion of the annealed complex formed by the adapter nucleic acid and the second nucleic acid may comprises a promoter. Both strands of the promoter may be DNA.
- the promoter may be a synthetic or naturally-derived molecule which is capable of conferring, activating or enhancing expression of a nucleic acid in a cell.
- the promoter may comprise one or more specific transcriptional regulatory sequences to further enhance expression and/or to alter the spatial expression and/or temporal expression of same.
- a promoter may also comprise distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
- a promoter may be derived from sources including viral, bacterial, fungal, plants, insects, and animals.
- a promoter may regulate the expression of a gene component constitutively, or differentially with respect to cell, the tissue or organ in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as physiological stresses, pathogens, metal ions, or inducing agents.
- promoters include the T7 promoter, T3 promoter, SP6 promoter, lac operator-promoter, tac promoter, S V40 late promoter, SV40 early promoter, RSV-LTR promoter, CMV JE promoter, S V40 early promoter or S V40 late promoter and the CMV JE promoter. 16.
- a method of detecting a target nucleic acid in a biological sample is also provided.
- the reverse-complement of the target nucleic acid may be synthesized.
- the target nucleic acid may then be detected in the biological sample by detecting binding of a probe comprising the target sequence to the synthesized reverse-complement. Binding of the probe to the reverse- compliment may be performed using a microarray or by Luminex analysis.
- the probe may be attached to a biochip.
- the probe may also comprise locked nucleic acids, which may increase the sensitivity of detection
- the ability to detect the target nucleic acid provides a wealth of information about the targeted nucleic acid, including whether the targeted nucleic acid is expressed in a particular cell, the level of the targeted nucleic acid expression in a particular cell or in comparison to other cells, identiBr the exact sequence of the targeted nucleic acid.
- Examples of methods based on the detection of a target nucleic acid, such as diagnostics and prognostics, are disclosed in U.S. Patent Application Nos. 10/536,560, 10/543,164, 11/130,645, 60/728,161 and 60/739,522; and International Patent Application Nos.
- the format for the genomic location is a concatenation of ⁇ chr_id> ⁇ strand> ⁇ start position>.
- 19+135460000 refers chromosome 19, +strand, start position 135460000.
- Chromosomes 23-25 refer to chromosome X, chromosome Y and mitochondrial DNA, respectively.
- the chromosomal location is based on the hgl7 assembly of the human genome by UCSC (http://genome.ucsc.edu), which is based on NCBI Build 35 version 1 and was produced by the International Human Genome Sequencing Consortium.
- Table 1 also lists whether the hairpin is conserved in evolution ("C”).
- the hairpins were identified as conserved ("Y”) or nonconserved (“N”) by using phastCons data.
- the phastCons data is a measure of evolutionary conservation for each nucleotide in the human genome against the genomes of chimp, mouse, rat, dog, chicken, frog, and zebrafish, based on a phylo-HMM using best-in-genome pair wise alignment for each species based on BlastZ, followed by multiZ alignment of the 8 genomes (Siepel et al, J. Comput. Biol 11, 413-428, 2004 and Schwartz et al., Genome Res. 13, 103-107, 2003).
- a hairpin is listed as conserved if the average phastCons conservation score over the 7 species in any 15 nucleotide sequence within the hairpin stem is at least 0.9 (Berezikov,E. et al. Phylogenetic Shadowing and Computational Identification of Human microRNA Genes. Cell 120, 21-24, 2005).
- Table 1 also lists the genomic type for each hairpin (“T”) as either intergenic (“G”), intron (“I”) or exon (“E”).
- Table 1 also lists the SEQ ID NO (“MID”) for each predicted miRNA and miRNA*.
- Table 1 also lists the prediction score grade for each hairpin (“P") on a scale of 0-1 (1 the hairpin is the most reliable), as described in Hofacker et al., Monatshefte f. Chemie 125: 167-188, 1994. If the grade is zero or null, they are transformed to the lower value of PalGrade that its p-value is ⁇ 0.05.
- Such undetected miRNAs may be expressed in tissues other than those tested. In addition, such undetected miRNAs may be expressed in the test tissues, but at a difference stage or under different condition than those of the experimental cells.
- D differentially expressed
- F Sanger DB Release 7.1
- Table 1 also lists a genetic location cluster ("LC”) for those hairpins that are within 5,000 nucleotides of each other. Each miRNA that has the same LC share the same genetic cluster. Table 1 also lists a seed cluster ("SC") to group miRNAs by their seed of 2-7 by an exact match. Each miRNA that has the same SC have the same seed. For a discussion of seed lengths of 5-6 nucleotides being sufficient for miRNA activity, see Lewis et al., Cell, 120;15-20 (2005).
- LC genetic location cluster
- SC seed cluster
- Table 5 lists the predicted target gene for each miRNA (MID) and its hairpin (HID) from the computational screen.
- the names of the target genes were taken from NCBI Reference Sequence release 9 (http://www.ncbi.nlm.nih.gov; Pruitt et al., Nucleic Acids Res, 33(l):D501- D504, 2005; Pruitt et al., Trends Genet., 16(l):44-47, 2000; and Tatusova et al., Bioinformatics, 15(7-8):536-43, 1999).
- positions 2--7 For a discussion on identifying target genes, see Lewis et al., Cell, 120: 15-20, (2005).
- the seed being sufficient for binding of a miRNA to a UTR, see Lim Lau et al., (Nature 2005) and Brenneck et al, (PLoS Biol 2005).
- Binding sites were then predicted using a filtered target genes dataset by including only those target genes that contained a UTR of a least 30 nucleotides.
- the binding site screen only considered the first 8000 nucleotides per UTR and considered the longest transcript when there were several transcripts per gene. A total of 14,236 transcripts were included in the dataset.
- Table 5 lists the SEQ ID NO for the predicted binding sites for each target gene as predicted from each miRNA ("MID"). The sequence of the binding site includes the 20 nucleotides 5' and 3' of the binding site as they are located on the spliced mRNA.
- Table 6 shows the relationship between the miRNAs ("MID”)/hairpins (“HID”) and diseases by their target genes.
- the name of diseases are taken from OMIM.
- Table 6 shows the number of miRNA target genes (“N") that are related to the disease.
- N miRNA target genes
- T total number of genes that are related to the disease
- P percentage of N out of T
- Pval p-value of hypergeometric analysis
- Table 10 shows the disease codes for the diseases described in Tables 6-7 and 11-12.
- Table 7 shows the relationship between the miRNAs ("MID")/hairpins ("HID”) and diseases by their host genes.
- MID miRNAs
- HID hairpins
- Intron_c Intron
- Exon_c Exon.
- two statuses like when Intron and Exon_c Intron is the one chosen.
- the logic of choosing is Intron>Exon>Intron_c>Exon_c>Intergenic.
- Table 11 shows the relationship between the target sequences ("Gene Name”) and disease ("Disease Code").
- Table 12 shows the relationship between the miRNAs ("MID”)/hairpms ("HID”), known SNPs and diseases.
- SNP were identified in the sequence of hairpins.
- the numeric code of the relevant diseases for each miRNA according to TablelO are presented in Table 12. The disease codes are taken from Table 10.
- Each SNP (“SNPJd ”) is identified based on NCBI database dbSNP BUILD 123 based on NCBI Human Genome Build 35.
- SNP_location The genomic location for each SNP
- SNP_location is also provided in a formation concatenating " ⁇ chr_id>: ⁇ start position>".
- “19:135460569” means chrl9 +strand, start position 135460569.
- SNPs a number of the mutations cover a few nucleotides (e.g., small insertions, deletions, micro-satellites, etc.)
- Swibertus (Blood 1996) and Frittitta (Diabetes 2001).
- Table 2 shows the hairpins ("HID") of the second prediction set that were validated by detecting expression of related miRNAs ("MID”), as well as a code for the tissue (“Tissue”) that expression was detected.
- the tissue and diseases codes for Table 2 are listed in Table 8.
- Some of the tested tissues were cell lines. Lung carcinoma cell line (H1299) with/without P53: H1299 has a mutated P53. The cell line was transfected with a construct with P53 that is temperature sensitive (active at 32°C). The experiment was conducted at 32°C.
- Table 2 also shows the chip expression score grade ("S”)(range of 500-65000). A threshold of 500 was used to eliminate non-significant signals and the score was normalized by MirChip probe signals from different experiments.
- Variations in the intensities of fluorescence material between experiments may be due to variability in RNA preparation or labeling efficiency.
- Each miRNA has an internal control of probes with mismatches.
- the relevant control group contained probes with similar C and G percentage (abs diff ⁇ 5%) in order to have similar Tm.
- the probe signal P value is the ratio over the relevant control group probes with the same or higher signals.
- the results are p-value -$.05 and score is above 500. In those cases that the SPVaI is listed as 0.0, the value is less than 0.0001.
- the data was obtained using a chip an internal control of probes with mismatches, which were checked for each significant signal that was affected in the mutated probes.
- the blots were dried and incubated overnight in separate hybridization bottles with 10 ml of ULTRAhyb-Oligo (Ambion) and 107 cpm of radio-labeled oligonucleotides complementary to the predicted miRNAs.
- the blots were washed 3x10 min at room temperature in 2x SSC, 0.5% SDS and then 1x15 min at 42 0 C in 2x SSC, 0.5% SDS.
- Overnight phosphorimaging using the Storm system (Amersham) revealed probes hybridizing to the predicted miRNAs.
- a group of the validated miRNAs from Example 3 were highly expressed in placenta, have distinct sequence similarity, and are located in the same locus on chromosome 19 ( Figure 2). These predicted miRNAs are spread along a region of ⁇ 100,000 nucleotides in the 19ql3.42 locus. This genomic region is devoid of protein-coding genes and seems to be intergenic. Further analysis of the genomic sequence, including a thorough examination of the output of our prediction algorithm, revealed many more putative related miRNAs, and located mir-371, mir-372, and mir-373 approximately 25,000bp downstream to this region. Overall, 54 putative miRNA precursors were identified in this region. The miRNA precursors can be divided into four distinct types of related sequences ( Figure 2).
- A-type miRNAs About 75% of the miRNAs in the cluster are highly related and were labeled as type A. Three other miRNA types, types B, C and D, are composed of 4, 2, and 2 precursors, respectively. An additional 3 putative miRNA precursors (Sl to S3) have unrelated sequences. Interestingly, all miRNA precursors are in the same orientation as the neighboring mir-371, mir-372, and mir-373 miRNA precursors.
- Sl to S3 putative miRNA precursors
- the repeating unit is almost always bounded by upstream and downstream AIu repeats. This is in sharp contrast to the MC 14-1 cluster which is extremely poor in AIu repeats.
- Figure 3-A shows a comparison of sequences of the 35 repeat units containing the A-type miRNA precursors in human. The comparison identified two regions exhibiting the highest sequence similarity. One region includes the A-type miRNA, located in the 3' region of the repeat. The second region is located -100 nucleotides upstream to the A-type miRNA precursors. However, the second region does not show high similarity among the chimp repeat units while the region containing the A-type miRNA precursors does ( Figure 3-B). [0143] Examination of the region containing the A-type repeats showed that the 5' region of the miRNAs encoded by the 5' stem of the precursors (5p miRNAs) seem to be more variable than other regions of the mature miRNAs.
- the multiple sequence alignment presented in Figure 4 revealed the following findings with regards to the predicted mature miRNAs.
- the 5p miRNAs can be divided into 3 blocks.
- Nucleotides 1 to 6 are C/T rich, relatively variable, and are marked in most miRNAs by a CTC motif in nucleotides 3 to 5.
- Nucleotides 7 to 15 are A/G rich and apart from nucleotides 7 and 8 are shared among most of the miRNAs.
- Nucleotides 16 to 23 are C/T rich and are, again, conserved among the members.
- the predicted 3p miRNAs in general, show a higher conservation among the family members. Most start with an AAA motif, but a few have a different 5' sequence that may be critical in their target recognition.
- Nucleotides 8 to 15 are C/T rich and show high conservation. The last 7 nucleotides are somewhat less conserved but include a GAG motif in nucleotides 17 to 19 that is common to most members.
- the two D-type miRNAs which are ⁇ 2000 nucleotides from each other, are located at the beginning of the cluster and are included in a duplicated region of 1220 nucleotides. Interestingly, the two D-type precursors are identical. Two of the three miRNAs of unrelated sequence, Sl and S2, are located just after the two D-type miRNAs, and the third is located between A34 and A35. In general, the entire ⁇ 100,000 nucleotide region containing the cluster is covered with repeating elements. This includes the miRNA-containing repeating units that are specific to this region and the genome wide repeat elements that are spread in the cluster in large numbers. EXAMPLE 5 Cloning Of Predicted MiRNAs
- Example 4 To further validate the predicted miRNAs, a number of the miRNAs described in Example 4 were cloned using methods similar to those described in U.S. Patent Application Nos. 60/522,459, 10/709,577 and 10/709,572, the contents of which are incorporated herein by reference. Briefly, a specific capture oligonucleotide was designed for each of the predicted miRNAs. The oligonucleotide was used to capture, clone, and sequence the specific miRNA from a placenta-derived library enriched for small RNAs.
- the 5' heterogeneity involved mainly addition of one nucleotide, mostly C or A, but in one case there was an addition of 3 nucleotides. This phenomenon is not specific to the miRNAs in the chromosome 19 cluster. We have observed it for many additional cloned miRNAs, including both known miRNAs as well as novel miRNAs from other chromosomes (data not shown).
- RT-PCR analysis was performed using 5 ⁇ g of placenta total RNA using oligo-dT as primer.
- the following primers were used to amplify the transcripts: fl : 5'-GTCCCTGTACTGGAACTTGAG-S'; f2: 5'-GTGTCCCTGTACTGGAACGCA-S'; rl: 5'-GCCTGGCCATGTCAGCTACG-S'; r2: 5'-TTGATGGGAGGCTAGTGTTTC-S'; r3: 5'-GACGTGGAGGCGTTCTTAGTC-S'; and r4: 5'-TGACAACCGTTGGGGATTAC-S'.
- the authenticity of the fragment was validated by sequencing.
- This region includes mir-A42 and mir-A43, which shows that both miRNAs are present on the same primary transcript.
- Further information on the transcription of the cluster came from analysis of the 77 ESTs located within it. We found that 42 of the ESTs were derived from placenta. As these ESTs are spread along the entire cluster, it suggested that the entire cluster is expressed in placenta. This observation is in-line with the expression profile observed in the microarray analysis. Thus, all miRNAs in the cluster may be co-expressed, with the only exception being the D-type miRNAs which are the only miRNAs to be expressed in HeLa cells. Interestingly, none of the 77 ESTs located in the region overlap the miRNA precursors in the cluster. This is in-line with the depletion of EST representation from transcripts processed by Drosha.
- MC 19-1 cluster is a natural part of chromosome 19.
- the MC14-1 cluster is generally conserved in mouse and includes only the A7 and A8 miRNAs within the cluster are not conserved beyond primates (Seitz 2004). In contrast all miRNAs in the MC19-1 cluster are unique to primates. A survey of all miRNAs found in Sanger revealed that only three miRNA, mir-198, mir-373, and mir-422a, are not conserved in the mouse or rat genomes, however, they are conserved in the dog genome and are thus not specific to primates.
- mir-371 and mir-372 which are clustered with mir- 373, and are located 25kb downstream to the MC19-1 cluster, are homologous to some extent to the A-type miRNAs (Figure 4), but are conserved in rodents.
- FIG. 4C Comparison of the A-type miRNA sequences to the miRNAs in the Sanger database revealed the greatest homology to the human mir-302 family ( Figure 4-C). This homology is higher than the homology observed with mir-371,2,3.
- the mir-302 family (mir-302a, b, c, and d) are found in a tightly packed cluster of five miRNAs (including mir-367) covering 690 nucleotides located in the antisense orientation in the first intron within the protein coding exons of the HDCMAl 8P gene (accession NM_ 016648). No additional homology, apart from the miRNA homology, exists between the mir-302 cluster and the MC 19-1 cluster. The fact that both the mir-371,2,3 and mir-302a,b,c,d are specific to embryonic stem cells is noteworthy.
- One of the predicted and validated human miRNA genes is the 13_12 gene (SEQ ID NO: IA), which is located at residues 14305325-14305407 of the + strand of chromosome 16, with reference to version HGl 7 of the human genome (References to SEQ ID NOS or MID numbers ending with "A” refer to the correspond label without the "A” in U.S. Patent Application No. 11/275,628, which are incorporated herein by reference).
- Figure 6 shows the predicted hairpin formed by the pre-miRNA (SEQ ID NO: 2A) encoded by 13_12, with the residues of the predicted miRNA and miRNA* indicated with underlining.
- the 13_12 gene (annotated as has-mir-193b at www.sanger.ac.uk) is homologous to the following predicted miRNAs: has-mir-193a (human), mmu-mir-193 (mouse), rno-mir-193 (rat), dre-mir-193a (zebra fish) and dre-mir-193b (zebra fish).
- An alignment of the genes encoding these miRNAs ( Figure 7) indicates that both the 5' and 3 1 mature miRNAs have high similarity and the 3' mature miRNAs have identical seeds, which may be important for target recognition.
- the physical location of the 13 12 gene is near has-mir-365-1 (has-mir-193a is also near has-mir-365-2).
- the 13_12 gene and has-mir-365-1 are apparently expressed from the same transcript based on reviewing the human mRNA and human EST databases using the UCSC Genome Browser.
- the coexpression of miRNAs on a single transcript has been previously reported (Baskerville et al., RNA, 11(3):241-7, 2005). CpG islands are also present near the beginning of the transcript.
- M A or C
- W A or U
- K G or U
- S C or G
- H A, C or U
- R A or G
- the miRNA inhibitors were used to test the effect on cell growth by inhibiting miRNA activity in PC-3 prostrate cancer cells. Although expression of 13__12 was not able to be detected in PC-3 cells using the methods described above, PC-3 prostate cancer cells were nonetheless used to assay the effects of the miRNA inhibitors because the detection methods used were not very sensitive.
- the inhibitors were tested by transfecting PC-3 cells in six replicate wells. For all transfections, wells contained 5,000-6,000 cells, 250 nM inhibitor and 0.3 ⁇ l LIPOFECTAMIN 2000 (#11668-019 Invitrogen). After 72 h the samples were assayed with MTT (CellTiter 96 non radioactive cell pro.
- the 13_12 gene is homologous to the gene encoding has-mir-193a.
- Inhibitors of has-mir-193a have been shown to cause a decrease of 50%-80% in the relative cell growth of A549 lung cancer cells. See Cheng et al., Nucleic Acids Res. 33(4):1290-7 (2005).
- A549 cells were transfected with inhibitors 193 and 13_12 and affects on cell growth measured using methods similar to those previously discussed in Example 2.
- the results in Figure 13 indicate that inhibition of 13_12 is at least as effective as inhibition of has-mir-193a in reducing proliferation of A549 lung cancer cells. These results are in contrast to the effect of the inhibitors in PC-3 cells discussed above, where the 193 inhibitor did not inhibit " cell growth.
- RNA is fractioned onto YM-100 columns to separate the small RNA molecules.
- the small RNA molecules are then precipitated in EtOH overnight at 40°C and resuspended in a buffer suitable for ligation of the resuspended RNA.
- An adaptor with SEQ ID NO: IB is then added to the mixture and ligation is carried on using T4 ligase (NEB) in ligase buffer including DMSO (15%)(References to SEQ ID NOS ending with "B” refer to the correspond label without the "B” in U.S. Provisional Patent Application No. 60/743,098, which are incorporated herein by reference).
- the reaction may be spiked with known RNA.
- the ligation products are then separated by electrophoresis in acrylamide gel. After the separation is completed, the ligation products are cut out from the gel and extracted out of the gel using GebaFlex electrophoration dialysis tubes. The extraction is followed by overnight precipitation in EtOH.
- a DNA oligo with SEQ ID NO: 2B is then annealed to T7 promoter to create a double stranded complex at the T7 promoter region. Annealing is controlled by step decrease of temperature from 70°C to 25°C, after 2 minutes at 85°C.
- Transcription is then performed using the Megashortscript kit (Ambion) for transcription of short sequences.
- the transcription can be carried on using biotinylated UTP and or biotynilated CTP to produce a labeled RNA transcript.
- the adaptor is then digested with DNase for 15 minutes at 37°C and final RNA transcript is then purified by chloroform/phenol phase extraction and EtOH precipitation overnight.
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